The long-term objective of our research is to understand how TRP activities are regulated. Recently, we identified a novel gene, called phosphoinositide interacting regulator of TRP (Pirt), which is highly expressed in pain-sensing, or nociceptive neurons in dorsal root ganglia (DRG) and absent in the central nervous system. Pirt encodes a two-transmembrane domain protein. Behavioral and electrophysiological studies of Pirt null mice suggest that Pirt functions as a positive regulator of TRPV1. Biochemical analysis shows that Pirt binds both TRPs and phosphatidylinositol-4,5-bisphosphate (PIP2). Importantly, Pirt enhances TRPV1 activity via PIP2. Our preliminary data suggest that Pirt can also bind and modulate TRPM8, a molecular sensor for cold sensation and menthol. In addition, we identified another structurally related PIP2 binding protein, called Pirt2. Interestingly, Pirt2 also binds certain TRPs including TRPA1 and TRPV1. In this proposal, we will take behavioral, electrophysiological, and biochemical approaches to further study the role of Pirt and Pirt2 in regulating these PIP2-sensitive TRP channels. Aim I is to assess whether the positive effect of Pirt on TRPM8 is also found in cultured DRG neurons by comparing neurons from wild-type and Pirt null mice. We will study how Pirt affects channel properties of TRPM8 at the single channel level. To determine whether Pirt affects TRPM8-mediated cold sensation in vivo, we will perform previously described behavioral tests on Pirt null mice and their wild-type littermates. In Aim II, we will study the molecular mechanism of how Pirt regulates TRPM8. We will employ biochemical approach to determine whether TRPM8 and Pirt form a complex in DRG neurons and which domains in Pirt and TRPM8 are required for their binding. Then we will assess whether Pirt and PIP2 require each other to enhance TRPM8. Aim III is to determine whether Pirt2 plays role in modulation of TRPA1 via PIP2. We will test the hypothesis that Pirt2 is involved in the PIP2 inhibitory effect on TRPV1. By analyzing the behavioral phenotypes of Pirt2 knockout mice, we will know whether Pirt2 plays role in TRPA1- and TRPV1-mediated pain sensation. The proposed analysis of Pirt and Pirt2 will facilitate our understanding of TRP regulation. PUBLIC HEALTH RELEVANCE: Transient receptor potential (TRP) channels are involved in many fundamental biological processes including pain sensation. Our studies suggest that Pirt and Pirt2 represent a novel family of TRP channel regulators. Therefore, functional analysis of Pirt and Pirt2 should facilitate our understanding of TRP modulation and thereby open the door for developing novel drugs to treat conditions like chronic pain.